Materials and methods for treatment of cancer and identification of anti-cancer compounds

ABSTRACT

The subject invention pertains to the treatment of tumors and cancerous tissues and the prevention of tumorigenesis and malignant transformation through the modulation of JAK/STAT3 intracellular signaling. The subject invention concerns pharmaceutical compositions containing cucurbitacin I, or a pharmaceutically acceptable salt or analog thereof, to a patient, wherein the tumor is characterized by the constitutive activation of the JAK/STAT3 intracellular signaling pathway. The present invention further pertains to methods of moderating the JAK and/or STAT3 signaling pathwaysin vitro or in vivo using cucurbitacin I, or a pharmaceutically acceptable salt or analog therof. Another aspect of the present invention concerns a method for screening candidate compoudns for JAK AND/or STAT3 inhibition and anti-tumor activity.

CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of provisional patentapplication Serial No. 60/279,104, filed Mar. 28, 2001.

[0002] The subject invention was made with government support under aresearch project supported by National Cancer Institute grant CA 78038.The Federal Government may have certain rights in this invention.

BACKGROUND OF INVENTION

[0003] The signal transducers and activators of transcription (STATs)are key signal transduction proteins that play a dual role oftransducing biological information from cell surface receptors to thecytoplasm and translocating to the nucleus where, as transcriptionfactors, they regulate gene expression (reviewed in Stark, G. R. et al.Annu. Rev. Biochem., 1998, 67:227-264; Horvath, C. M. and J. E. DarnellCurr. Opin. Cell. Biol., 1997, 9:233-239, Ihle, J. N. and I. M. KerrTrends Genet., 1995, 11:69-74; Schindler, C. and J. E. Darnell Annu.Rev. Biochem., 1995, 64:621-651). Mammalian cells express sevendifferent STATs (1, 2, 3, 4, 5a, 5b, and 6). Gene knockout and otherexperiments implicated STATs in many important physiological functionssuch as immune modulation, inflammation, proliferation, differentiation,development, cell survival and apoptosis (Stark, G. R. et al. Annu. Rev.Biochem., 1998, 67:227-264; Horvath, C. M. and J. E. Darnell Curr. Opin.Cell Biol., 1997, 9:233-239, Ihle, J. N. and I. M. Kerr Trends Genet.,1995, 11:69-74; Schindler, C. and J. E. Darnell Annu. Rev. Biochem.,1995, 64:621-651).

[0004] STAT tyrosine phosphorylation is required for their biologicalfunction. This occurs when cytokines such as interleukin-6 andinterferon or growth factors such as PDGF and EGF bind their respectivereceptors which results in STAT protein recruitment to the inner surfaceof the plasma membrane in the vicinity of the cytoplasmic portion of thereceptors (Ihle, J. N. et al. Annu. Rev. Immunol., 1995, 13:369-398;Leaman, D. W. et al. Faseb J., 1996, 10:1578-1588). Tyrosine kinasesthat are known to phosphorylate STATs are non-receptor tyrosine kinasessuch as Src and the Janus kinases, JAK1 and JAK2. Other possibletyrosine kinases that can phosphorylate STATs are peptide growth factorreceptors such as PDGFR and EGFR. The cellular levels of STATs that aretyrosine phosphorylated could also be regulated by phosphotyrosine STATphosphatases such as SHP-1 and SHP-2 (Schaper, F. et al. Biochem J.,1998, 335:557-565; Stofega, M. R. et al. J. Biol. Chem., 1998,273:7112-7117; Yu, C. L. et al. J. Biol. Chem., 2000, 275:599-604). Oncetyrosine phosphorylated, STAT monomers dimerize via reciprocalphosphotyrosine-SH2 interactions and translocate to the nucleus wherethey bind DNA and regulate gene transcription (Ihle, J. N. and I. M.Kerr Trends Genet., 1995, 11:69-74; Seidel, H. M. et al. Proc. Natl.Acad. Sci. USA, 1995, 92:3041-3045). Whereas tyrosine phosphorylation ofSTATs regulates dimerization, nuclear translocation and DNA-binding,serine/threonine phosphorylation is believed to regulate thetranscriptional activity of STATs (Turkson, J. et al. Mol. Cell Biol.,1999, 19:7519-7528).

[0005] Several lines of evidence have implicated some STAT familymembers in malignant transformation and tumor cell survival (Bowman, T.et al. Cancer Control, 1999, 6:427-435; Turkson, J and R. Jove Oncogene,2000, 19:6613-6626). STAT3 involvement in oncogenesis is the mostthoroughly characterized. First, STAT3 is found constitutively tyrosinephosphorylated and activated in many human cancers (Bowman, T. et al.Cancer Control, 1999, 6:427-435; Turkson, J and R. Jove Oncogene, 2000,19:6613-6626; Bowman, T. et al. Oncogene, 2000, 19:2474-2488). Thisabnormal activation of STAT3 is prevalent in breast, pancreas, ovarian,head and neck, brain, and prostate carcinomas as well as melanomas,leukemias and lymphomas. In those tumors investigated, aberrant STAT3activation is required for growth and survival (Bowman, T. et al. CancerControl, 1999, 6:427-435; Turkson, J and R. Jove Oncogene, 2000,19:6613-6626; Bowman, T. et al. Oncogene, 2000, 19:2474-2488). Second,many known oncogenes, especially those belonging to the non-receptortyrosine kinase family such as src, induce constitutive activation ofSTAT3 (Yu, C. L. et al. Science, 1995, 269:81-83). Third, expression ofa constitutively-activated mutant of STAT3, where stable dimerizationwas forced through disulfide covalent linkage, was shown to besufficient to induce cell transformation and tumor growth in nude mice(Bromberg, J. F. et al. Cell, 1999, 98:295-303). Finally, perhaps themost compelling evidence for the requirement of STAT3 for oncogenesisand its validation as an anticancer drug target comes from experimentswhere a dominant negative form of STAT3 was used in cultured cells aswell as in gene therapy animal experiments to show that blockingaberrant activation of STAT3 results in inhibition of tumor growth andsurvival and induction of apoptosis with little side effects to normalcells (Niu, G. et al. Cancer Res., 1999, 59:5059-5063; Catlett-Falcone,R. et al. Immunity, 1999, 10:105-115).

[0006] Much of modern anticancer drug discovery approaches have focusedon targeting signal transduction pathways involving receptor tyrosinekinases (e.g., ErbB2, EGFR), farnesylated proteins (e.g., Ras), andnon-receptor cytosolic kinases (e.g. Raf, Mek, PI3K, and Akt) (Sebti, S.New Drug Targets and Therapies for Cancer, 2000, 6549-6692). Theseimportant efforts resulted in several novel agents such as RTKmonoclonal antibodies and RTK, farnesyltransferase, Raf, and Mekinhibitors that are presently in clinical trials, such as the Bcr-Abltyrosine kinase inhibitor STI-571 (GLEEVEC), which has recently beenapproved by the FDA for chronic myelogenous leukemia.

[0007] In contrast to the heavily exploited area described above, littlehas been done to target the STAT3 signaling pathway. Yet, experiments inanimal models using gene therapy with a dominant negative form of STAT3and a constitutively-active mutant of STAT3, as well as the prevalenceof constitutively-activated STAT3 in many human cancers, stronglysuggest that STAT3 has a causal role in oncogenesis (Bowman, T. et al.Cancer Control, 1999, 6:427-435; Turkson, J and R. Jove Oncogene, 2000,19:6613-6626; Bowman, T. et al. Oncogene, 2000, 19:2474-2488).Furthermore, the fact that constitutive activation of STAT3 inducesgenes such as cyclin D1, c-myc, and bcl-xl that are intimately involvedin oncogenesis and tumor survival, coupled with the fact thatconstitutively-activated STAT3 is required for survival of some humancancer cells, further validates the STAT3 signaling pathway as aselective cancer drug discovery target (Bowman, T. et al. CancerControl, 1999, 6:427-435; Turkson, J and R. Jove Oncogene, 2000,19:6613-6626; Bowman, T. et al. Oncogene, 2000, 19:2474-2488.

[0008] Based on the observations described above, some researchers haveundertaken to target STAT3 for the development of novel anti-cancerdrugs (reviewed in Bowman, T. et al. Cancer Control, 1999, 6:427-435;Turkson, J and R. Jove Oncogene, 2000, 19:6613-6626; and Bowman, T. etal. Oncogene, 2000, 19:2474-2488). Depending on the aberrant geneticalterations that result in constitutively tyrosine-phosphorylated,activated STAT3, several approaches can be undertaken including blockingligand/receptor interactions, inhibiting receptor and non-receptortyrosine kinases, activating phosphotyrosine STAT3 phosphatases, andblocking STAT3 dimerization, nuclear translocation, DNA-binding, andgene transcription. In addition, gene therapy, anti-sense, or RNAiapproaches can also be attempted.

[0009] JSI-124 is a plant natural product previously identified ascucurbitacin I, a member of the cucurbitacin family of compounds thatare isolated from various plant families, such as the Cucurbitaceae andCruciferae, and that have been used as folk medicines for centuries incountries such as China and India. However, little was known about thebiological activities of the various cucurbitacins until recently. Somecucurbitacins have been shown to have anti-inflammatory and analgesic aswell as cytotoxic effects. Furthermore, cucurbitacins were also found toinhibit DNA, RNA and protein synthesis in HeLa cells (Witkowski, A. etal. Biochem Pharmacol, 1984, 33:995-1004) and inhibit proliferation ofHeLa cells (Witkowski, A. et al. Biochem Pharmacol, 1984, 33:995-1004),endothelial cells (Duncan, M. D. and K. L. Duncan J. Surg. Res., 1997,69:55-60) and T-lymphocytes (Smit, H. F. et al. J. Nat. Prod., 2000,63:1300-1302). Finally, some cucurbitacins were shown to suppress skincarcinogenesis (Konoshima, T. et al. Biol. Pharm. Bull., 1995,18:284-287), inhibit cell adhesion (Musza, L. L. et al. J. Nat. Prod.,1994, 57:1498-1502) and disrupt the actin and vimentin cytoskeleton inprostate carcinoma cells (Duncan, K. L. et al. Biochem Pharmacol, 1996,52:1553-1560; Duncan, M. D. and K. L. Duncan J. Surg. Res., 1997,69:55-60).

BRIEF SUMMARY OF THE INVENTION

[0010] The subject invention relates to the identification of substancescapable of interfering with the signaling events leading to theabnormally elevated levels of tyrosine phosphorylated STAT3 in manyhuman cancer cells. More specifically, the subject invention concernsthe identification of substances that act as inhibitors of STAT3activation pathways. The subject invention further concerns thetreatment of tumors and cancerous tissues and the prevention oftumorigenesis and malignant transformation through the disruption ofSTAT3 intracellular signaling.

[0011] In one aspect, the subject invention concerns a pharmaceuticalcomposition comprising a compound having the structure shown in FIG. 1(also referred to herein as JSI-124 or cucurbitacin I), which is apotent suppressor of the JAK/STAT3 tumor survival pathway, and whichexhibits potent antitumor activity. In another aspect, the subjectinvention concerns a pharmaceutical composition comprising an analog ofcucurbitacin I, such as cucurbitacin A, cucurbitacin B, cucurbitacin D,cucurbitacin E, cucurbitacin Q, or tetrahydro-cucurbitacin I. Thepharmaceutical compositions of the subject invention are useful fortreating cancer and inhibiting tumor growth, wherein the cancer or tumoris characterized by constitutive activation of the JAK2 and/or STAT3signaling pathways.

[0012] The subject invention also concerns articles of manufactureuseful in treating cancer and inhibiting tumor growth, wherein thecancer or tumor is characterized by constitutive activation of the JAK2and/or STAT3 signaling pathways.

[0013] In another aspect, the subject invention concerns a method ofinhibiting the growth of cancer cells in a patient by the administrationof cucurbitacin I (JSI-124), or analogs thereof. In a further aspect,the present invention concerns methods for modulating STAT3 activity invitro or in vivo by administering cucurbitacin I, or analogs thereof.

[0014] In a further aspect, the subject invention concerns a method ofscreening substances for antitumor activity using a phosphotyrosineSTAT3-specific cytoblot. Using the screening method of the subjectinvention, modulation of STAT3 can be utilized to evaluate the antitumorefficacy of a candidate substance on a broad spectrum of cancer celllines.

[0015] In another aspect, the subject invention concerns a kit forscreening substances for antitumor activity comprising cells and aligand specific for phosphotyrosine-STAT3 protein, wherein the ligand isdirectly or indirectly associated with a detectable label. Optionally,the kit can further comprise a substrate for carrying out the assay.

BRIEF DESCRIPTION OF THE DRAWINGS

[0016]FIG. 1 shows the chemical structure of JSI-124 (cucurbitacin I),which was identified from the NCI diversity set using a phosphotyrosineSTAT3 high throughput cytoblot assay.

[0017]FIG. 2 shows v-Src-transformed NIH 3T3 cells containingconstitutively-activated tyrosine phosphorylated STAT3, which wereplated in all wells of the 96-well plate except for wells 1A, 1B, 1C and1D, where NIH 3T3 cells transfected with empty vector were plated. WellsA through H of column 1 were treated for 4 hours with vehicle, whereasall other wells were treated with compounds from the NCI diversity set(each well received a different compound). The cells were thenpermeabilized and cytoblotted with an anti-phosphotyrosineSTAT3-specific antibody as described under Materials and Methods.

[0018] FIGS. 3A-3C suppression of phosphotyrosine STAT3 levels inv-src-transformed NIH 3T3 cells and human cancer cell lines by JSI-124.FIG. 3A shows phosphotyrosine STAT3 levels in various human cell lines.Lysates from a variety of human cancer cell lines were processed forWestern blotting using anti-phosphotyrosine STAT3 antibody as describedin Materials and Methods. FIG. 3B shows suppression of phosphotyrosineSTAT3 levels by JSI-124. v-Src-transformed NIH 3T3 cells, A549 (a lungadenocarcinoma), MDA-MB-231 (a breast carcinoma), MDA-MB-468 (a breastcarcinoma) and Panc-1 (a pancreatic carcinoma) cells were treated for 4hours with either vehicle or JSI-124 (10 μM), harvested and processedfor anti-phosphotyrosine STAT3 Western blotting as described for 2a.FIG. 3C shows suppression of phosphotyrosine STAT3 levels by JSI-124without affecting STAT3 protein levels. A549 and MDA-MB-468 cells weretreated as in FIG. 3B except that the lysates were firstimmunoprecipitated with anti-STAT3 antibody and immunoblotted eitherwith anti-phosphotyrosine STAT3 antibody or anti-STAT3 antibody. Dataare representative of three independent experiments for FIGS. 3A, 3B,and 3C.

[0019]FIGS. 4A and 4B show inhibition of STAT3 DNA-binding activity andSTAT3-mediated transcription by JSI-124. In FIG. 4A, v-Src-transformedNIH 3T3 cells and A549 cells were treated with vehicle or JSI-124,harvested and processed for EMSA as described under Materials andMethods. Samples in lanes 1 through 4 and 6 through 9 are from cellsthat were treated with vehicle control, whereas samples from lanes 5 and10 are from cells treated with JSI-124. Lanes 2 and 3 as well as 7 and 8are from samples supershifted with anti-STAT1 or anti-STAT3 antibodies,respectively. In FIG. 4B, v-Src-transformed NIH 3T3 cells transfectedeither with STAT3 (pLucTKS3)- or SRE (pRLSRE)-dependent luciferasereporters were either treated with vehicle or JSI-124 and processed forluciferase assays as described under Materials and Methods. Data arerepresentative of two independent experiments.

[0020] FIGS. 5A-5C show the effects of JSI-124 on phosphotyrosine levelsand kinase activities of JAK and Src kinases. FIG. 5A shows suppressionof phosphotyrosine levels of STAT3 and JAK2 but not Src, by JSI-124.A549 and MDA-MB-468 cells were treated with various concentrations ofJSI-124 and processed for immunoblotting with antibodies specific foreither phosphotyrosine STAT3, phosphotyrosine JAK2 or phosphotyrosineSrc as described under Methods. The membranes were also reblotted withantibodies to STAT3 and JAK2. FIG. 5B shows rapid suppression by JSI-124of phosphotyrosine STAT3 and JAK2. A549 and MDA-MB-468 cells weretreated with JSI-124 (10 μM) for various lengths of time (0-240 min) andprocessed as described above. FIG. 5C shows that JSI-124 does notinhibit JAK1, JAK2 and Src kinase activities. Lysates from v-Srctransformed cells, A549 cells and MDA-MB-468 cells wereimmunoprecipitated with antibodies against JAK1, JAK2 and Src.Autophosphorylation kinase assays were then performed as described underMaterials and Methods. Immunoprecipitates were incubated either withvehicle control (C), JSI-124 (J), the JAK kinase inhibitor AG490 (A) orthe Src kinase inhibitor PD180970 (P). Data are representative of threeindependent experiments.

[0021]FIGS. 6A and 6B show suppression by JSI-124 of phosphorylationlevels of STAT3, but not Erk1/Erk2, JNK and Akt. A549 and MDA-MB-468were treated with various concentrations of JSI-124 (0-10 μM andprocessed for immunoblotting with phospho-specific antibody againsteither STAT3, Erk1, Erk2, JNK or Akt as described under Methods. Dataare representative of three independent experiments.

[0022] FIGS. 7A-7G show that JSI-124 increases mouse survival andinhibits tumor growth in mice of MDA-MB-468 (FIG. 7A), A-549 (FIG. 7B),B16-F10 (FIG. 7E) and v-Src/NIH 3T3 (FIG. 7D), but not Calu-1 (FIG. 7C)and Ras/NIH 3T3 cells (FIG. 7F). MDA-MB-468, A549, Calu-1, v-Src/NIH3T3, and Ras/NIH 3T3, cells were implanted s.c. in nude mice, whereasB316-F10 cells were implanted s.c. in C57 black mice. When the tumorsreached an average size of about 100 m³-150 mm³, animals were randomized(five animals per group; two tumors per animal) and treated with eithervehicle (circle) or 1 mg/kg/day JSI-124 (triangle) as described underMaterials and Methods. Each measurement represents an average of tentumors. Data are representative of four (B16-F10), three (A549,v-Src/NIH 3T3, and Ras/NIH 3T3) and two (MDA-MB-468 and Calu-1)independent experiments († designate P<0.01; **, P<0.05; *, P=0.08). Forthe mouse survival studies, C57 black mice were implanted s.c. withB16-F10 cells and on day 5 after implantation, the mice were randomized(6 animals per group) and treated with either vehicle (circles) orJSI-124 (triangles) (1 mg/Kg/day) for 25 days. Percent surviving micewas determined by monitoring the death of mice over a period of 42 daysuntil all mice died. Data are representative of two independentexperiments carried out with 12 mice each (6; vehicle control-treatedand 6; JSI-124-treated). For statistical analysis: for each of the 2experiments, control animals were compared to JSI-124 animals withrespect to survival using the permutation log rank test as implementedin the statistical software package, ProcStatXact. The results of bothexperiments were pooled in a stratified analysis and gave a p-value of0.01.

[0023]FIG. 8 shows chemical structure I, representing cucurbitacinanalogs of the subject invention.

[0024]FIG. 9 shows the chemical structure of cucurbitacin A, acucurbitacin analog of the subject invention.

[0025]FIG. 10 shows the chemical structure of cucurbitacin B, acucurbitacin analog of the subject invention.

[0026]FIG. 11 shows the chemical structure of cucurbitacin D, acucurbitacin analog of the subject invention.

[0027]FIG. 12 shows the chemical structure of cucurbitacin E, acucurbitacin analog of the subject invention.

[0028]FIG. 13 shows the chemical structure of cucurbitacin Q, acucurbitacin analog of the subject invention.

[0029]FIG. 14 shows the chemical structure of tetrahydro-cucurbitacin I,a cucurbitacin analog of the subject invention.

[0030]FIG. 15 shows representative blots of the effects of cucurbitacinsA, B, D, E, I, Tetrahydro-I, and Q (0 μM-10 μM) on the levels of phosphoSTAT3 and phospho JAK2 in A-549 cells.

[0031] FIGS. 16A-16C compare the ability of the cucurbitacin analogs toinhibit phospho STAT3 or phospho JAK2 levels to their ability to inhibittumor growth in nude mice. FIG. 16 shows that cucurbitacin I, whichinhibits both phospho STAT3 and phospho JAK2 levels, inhibits tumorgrowth. In contrast, cucurbitacin A, which inhibits only phospho JAK2,but no phospho STAT3 levels, did not significantly inhibit tumor growth.Cucurbitacin Q which inhibits phospho STAT3 but not phospho JAK2 levelswas as potent as cucurbatacin I at inhibiting tumor growth.

[0032]FIG. 17 is a table showing that cucurbitacin Q, tetrahydrocucurbitacin I, cucurbitacin I, and cucurbitacin E inhibit tumor growth,whereas curbitacin A did not and cucurbitacin B was toxic at 1mg/kg/day. At 200 μg/kg/day, cucurbitacin B was not toxic.

BRIEF DESCRIPTION OF THE SEQUENCES

[0033] SEQ ID NO. 1 is a ³²P-radiolabeled oligonucleotide probe thatbinds STAT1 and STAT3.

DETAILED DISCLOSURE OF THE INVENTION

[0034] The subject invention pertains to compounds capable ofinterfering with the signaling events leading to the abnormally elevatedlevels of tyrosine phosphorylated STAT3 in many human cancers.

[0035] In one aspect, the subject invention concerns a pharmaceuticalcomposition comprising a compound having the structure shown in FIG. 1(also referred to herein as JSI-124 or cucurbitacin I), which is apotent suppressor of the JAK/STAT3 tumor survival pathway, and whichexhibits potent antitumor activity. In another aspect, the presentinvention concerns a pharmaceutical composition comprising an analog ofcucurbitacin I. In a further aspect, the present invention concerns apharmaceutical composition comprising an analog of curbitacin I selectedfrom the group consisting of cucurbitacin A, cucurbitacin B,cucurbitacin D, cucurbitacin E, cucurbitacin Q, andtetrahydro-cucurbitacin I. The chemical structures of the cucurbitacinanalogs are shown in FIGS. 9-14, respectively.

[0036] In another aspect, the subject invention concerns a method ofinhibiting the growth of cancer cells in a patient by the administrationof cucurbitacin I (JSI-124), or analogs thereof. The method of thesubject invention is useful in treating cancer and inhibiting tumorgrowth, wherein the cancer or tumor is characterized by constitutiveactivation of the JAK2 and/or STAT3 signaling pathways. Treatment ofcancer involves a decrease of one or more symptoms associated with theparticular cancer. Preferably, the treatment involves a decrease intumor growth rate, particularly where the tumor is characterized byconstitutive activation of the JAK2 and/or STAT3 signaling pathways.

[0037] The present inventors have demonstrated that JSI-124 and analogsof JSI-124 reduce the levels of phosphotyrosine constitutively-activatedSTAT3 in many human cancer cell lines including pancreatic, lung andbreast carcinomas. Without being bound by theory, this suppression inthe levels of constitutively-activated STAT3 results in blockade ofSTAT3 DNA-binding activity and STAT3-mediated gene transcription.JSI-124 was highly selective for disrupting STAT3 signaling over otherpivotal oncogenic and tumor survival pathways. For example, in two celllines, the human lung adenocarcinoma A549 and the human breast carcinomaMDA-MB-468, JSI-124 did not inhibit the constitutive activation of theSer/Thr protein kinase B, PKB/Akt, indicating that thephosphoinositide-3-kinase (PI3K)/Akt survival pathway is not a targetfor JSI-124. Similarly, the Ras/Raf/Mek/Erk oncogenic signaling pathwaywas not inhibited by JSI-124 in these two cell lines. Finally, theconstitutive activation of Jun kinase in A549 and MDA-MB-468 was notaffected by JSI-124, indicating that the stress activated protein kinasesignaling pathway is not targeted by JSI-124.

[0038] A large number of human cancers rely on the PI3K/Akt and theRas/Raf/Mek/Erk pathways to induce malignant transformation and tumorsurvival. For example, the great majority of tumors overexpress the ErbBfamily of receptors such as EGFR and ErbB2 and contain mutant forms ofRas. These RTK and Ras genetic alterations result in constitutiveactivation of the PI3K/Akt and Ras/Raf/Mek/Erk pathways. The fact thatJSI-124 inhibits tumor growth and blocks STAT3 signaling withoutinhibiting the constitutive activation of Akt and Erk1/Erk2 suggeststhat its ability to inhibit the growth of A549 and MDA-MB-468 in nudemice does not depend on blocking Akt and Erk activation. This alsosuggests that JSI-124 may be more selective towards inhibiting thegrowth of tumors with constitutively-activated STAT3. Consistent withthis, it was found that v-Src-transformed NIH 3T3 tumors which depend onconstitutively-activated STAT3 for malignant transformation aresensitive to JSI-124 antitumor activity in nude mice. In contrast,oncogenic Ras-transformed NIH 3T3 tumors, where STAT3 is notconstitutively-activated, were resistant to JSI-124. Furthermore, thefact that JSI-124 inhibited the growth in mice of the human lungadenocarcinoma (A-549), the human breast carcinoma (MDA-MB-468) and themurine melanoma (B16-F10) all of which express constitutively activatedSTAT3 but not the human lung adenocarcinoma (Calu-1) that has very lowlevels of tyrosine phosphorylated STAT3 gives further support to thenotion that the ability of JSI-124 to inhibit tumor growth depends on anaberrantly activated STAT3 signaling pathway. Importantly, JSI-124 alsosignificantly increased the survival of immunologically-competent miceimplanted with B16-F10 murine melanoma.

[0039] The ability of JSI-124 to suppress the cellular levels ofphosphotyrosine-STAT3 but not phospho-Erk1/2, phospho-JNK, andphospho-Akt suggested that a STAT3 tyrosine kinase is a possiblemolecular target for JSI-124. Consistent with a direct inhibition of theenzymatic activity of a tyrosine kinase is the fact that suppression ofthe STAT3 phosphotyrosine levels was rapid (observed as early as 30 minand complete after only 2 hr of treatment). There are twowell-characterized STAT3 tyrosine kinases: JAK and Src kinase. Becausephosphotyrosine JAK2 levels were also reduced by JSI-124 this suggestedthat JAK2 is likely not the target. This was confirmed by in vitrokinase assays where JAK2 and JAK1 enzymatic activities were inhibited byAG490, a known JAK inhibitor, but not JSI-124. Similarly, Src kinaseactivity was inhibited in vitro by the known Src kinase inhibitorPD180970 but not JSI-124, indicating that Src kinase is not a target.The receptor tyrosine kinase EGFR that is believed also to phosphorylateSTAT3 is most likely not a target either since EGF-stimulation of EGFRtyrosine phosphorylation in the breast cell line MCF-10A andEGFR-overexpressing NIH 3T3 cells was inhibited only minimally byJSI-124 (data not shown).

[0040] Without being bound by theory, reduction in phosphotyrosinelevels could be due either to inhibition of protein tyrosine kinases oractivation of protein phosphotyrosine phosphatases (PTPs). STAT3 isknown to be phosphotyrosine dephosphorylated by two PTPs, SHP-1 andSHP-2 (Schaper, F. et al. Biochem. J., 1998, 335:557-565; Yu, C. L. etal. J. Biol. Chem., 2000, 275:599-604), and JSI-124 could downregulatephosphotyrosine-STAT3 levels by promoting the protein phosphataseactivities of SHP-1 and SHP-2. Alternatively, JSI-124 could alsoactivate physiological inhibitors that are known to directly orindirectly downregulate STAT3 activation. These include suppressors ofcytokine signaling (SOCS), STAT-induced STAT inhibitor (SSI), JAKbinding protein (JAB), and STAT3 interacting protein (StP1) (Turkson, J.and R. Jove Oncogene, 2000, 19:6613-6626).

[0041] According to the method of the subject invention, cucurbitacin I,or a pharmaceutically acceptable salt or analog thereof, is administeredto a patient in an effective amount to decrease the constitutive levelsof JAK2 activity, constitutive levels of STAT3 activity, or constitutivelevels of both JAK2 and STAT3 activity. The cucurbitacin I, or apharmaceutically acceptable salt or analog thereof, can be administeredprophylactically before tumor onset, or as treatment for existingtumors.

[0042] In one embodiment of the method of the present invention,cucurbitacin I, or a pharmaceutically acceptable salt or analog thereof,is administered to a patient, wherein the cucurbitacin analogadministered exhibits inhibition toward both JAK2 and STAT3 constitutiveactivity. In another embodiment of the method of the subject invention,a cucurbitacin analog is administered to a patient, wherein thecucurbitacin analog administered exhibits inhibition toward constitutiveSTAT3 activity, but not constitutive JAK2 activity. Preferably, thepatient is suffering from a form of cancer that is characterized byconstitutive activation of only the JAK2 signaling pathway, byconstitutive action of only the STAT3 signaling pathway, or byconstitutive action of both the JAK2 and STAT3 signaling pathway.

[0043] Cucurbitacin analogs can be used according to the methods of thesubject invention so long as the analogs exhibit the desired biologicalactivity. Biological activity characteristics can be evaluated using ahigh-throughput system, such as the method of the subject invention (thecytoblot assay), described in detail below, or other methods disclosedherein and/or known to those of ordinary skill in the art.

[0044] A cucurbitacin analog having the capability to modulate the JAKand/or STAT3 signaling pathway would be considered to have the desiredbiological activity in accordance with the subject invention. Fortherapeutic applications, an analog of the subject invention preferablyhas the capability to inhibit activation of the JAK and/or STAT3signaling pathway. Inhibition of STAT3 signaling by cucurbitacin I, andanalogs thereof, selectively promotes apoptosis in tumor cells thatharbor constitutively activated STAT3. Therefore, the desirable goals ofpromoting apoptosis (“programmed cell death”) of selective cancerouscells and suppression of malignant transformation of normal cells withina patient are likewise accomplished through administration ofantagonists or inhibitors of STAT 3 signaling of the present invention,which can be administered as simple compounds or in a pharmaceuticalformulation.

[0045] The cucurbitacin analogs of the subject invention includenaturally occurring analogs of cucurbitacin I and synthetic analogs ofcucurbitacin I. Cucurbitacin analogs of the subject invention can besubstituted at various positions. FIG. 8 shows a chemical structure(structure I) representing cucurbitacin analogs of the subjectinvention.

[0046] Referring to each of the chemical structures shown in FIG. 8(structure I). R₁, R₂, R₃, R₄, R₅, and R₆ can each be the same ordifferent. R₁, R₂, R₃, R₄, R₅, and R₆ can each be H, O, hydroxyl, alkyl,alkenyl, alkynyl, halogen, alkoxy, aryl, or heteroaryl.

[0047] As used in the specification, the term “alkyl” refers to astraight or branched chain alkyl moiety. In one embodiment, the alkylmoiety is C₁₋₈ alkyl, which refers to an alkyl moiety having from one toeight carbon atoms, including for example, methyl, ethyl, propyl,isopropyl, butyl, tert-butyl, pentyl, hexyl, octyl, and the like. Inanother embodiment, the alkyl moiety is C₁₋₃ alkyl.

[0048] The term “alkenyl” refers to a straight or branched chain alkylmoiety having in addition one or more carbon-carbon double bonds, ofeither E or Z stereochemistry where applicable. In one embodiment, thealkenyl moiety is C₂₋₆ alkenyl, which refers to an alkenyl moiety havingtwo to six carbon atoms. This term would include, for example, vinyl,1-propenyl, 1- and 2-butenyl, 2-methyl-2-propenyl, and the like.

[0049] The term “alkynyl” refers to a straight or branched chain alkylmoiety having in addition one or more carbon-carbon triple bonds. In oneembodiment, the alkynyl moiety is C₂₋₆ alkynyl, which refers to analkynyl moiety having two to six carbon atoms. This term would include,for example, ethynyl, 1-propynyl, 1- and 2-butynyl, 1-methyl-2-butynyl,and the like.

[0050] The term “alkoxy” refers to an alkyl-O-group, in which the alkylgroup is as previously described.

[0051] The term “halogen” refers to fluorine, chlorine, bromine, oriodine.

[0052] The term “aryl” refers to an aromatic carbocyclic ring,optionally substituted with, or fused with, an aryl group. This termincludes, for example, phenyl or napthyl.

[0053] The term “heteroaryl” refers to aromatic ring systems of five toten atoms of which at least one atom is selected from O, N, and S, andoptionally substituted with an aryl group substituent. This termincludes for example furanyl, thiophenyl, pyridyl, indolyl, quinolyl,and the like.

[0054] The term “aryl group substituent” refers to a substituent chosenfrom halogen, CN, CF₃, CH₂F, and NO₂.

[0055] The term “optionally substituted” means optionally substitutedwith one or more of the groups specified, at any available position orpositions.

[0056] It will be appreciated that the cucurbitacin analogs of thesubject can contain one or more asymmetrically substituted carbon atoms(chiral centers). The presence of one or more of these asymmetriccenters in an analog of the chemical structure shown in FIG. 8 can giverise to stereoisomers, and in each case the invention is to beunderstood to extend to all such stereoisomers, including enantiomersand diastereomers, and mixtures including racemic mixtures thereof,having JAK and/or STAT3 pathway inhibitory activity.

[0057] The precise dosage will depend on a number of clinical factors,for example, the type of patient (such as human, non-human mammal, orother animal), age of the patient, and the particular cancer undertreatment and its aggressiveness. A person having ordinary skill in theart would readily be able to determine, without undue experimentation,the appropriate dosages required to achieve the appropriate clinicaleffect.

[0058] A “patient” refers to a human, non-human mammal, or other animalin which inhibition of the JAK/STAT signaling pathway would have abeneficial effect. Patients in need of treatment involving inhibition ofthe JAK/STAT signaling pathway can be identified using standardtechniques known to those in the medical profession.

[0059] The compounds of the subject invention, including cucurbitacin I,and analogs thereof, can be obtained through a variety of methods knownin the art. For example, cucurbitacin I can be isolated and purifiedfrom various plant families, such as the Cucurbitaceae and Cruciferae.Cucurbitacin analogs of the subject invention can be synthesized usingmethods of organic synthesis known to those of ordinary skill in theart.

[0060] A further aspect of the present invention provides a method ofmodulating the activity of the JAK/STAT signaling pathway and includesthe step of contacting cells or tissue with cucurbitacin I, or analogsthereof, inhibiting activity of the JAK/STAT signaling pathway. Themethod can be carried out in vivo or in vitro.

[0061] While cucurbitacin I and cucurbitacin analogs can be administeredas an isolated compound, it is preferred to administer these compoundsas a pharmaceutical composition. The subject invention thus furtherprovides pharmaceutical compositions comprising cucurbitacin I, or ananalog thereof, as an active agent, or physiologically acceptablesalt(s) thereof, in association with at least one pharmaceuticallyacceptable carrier or diluent. The pharmaceutical composition can beadapted for various routes of administration, such as enteral,parenteral, intravenous, intramuscular, topical, subcutaneous, and soforth. Administration can be continuous or at distinct intervals, as canbe determined by a person of ordinary skill in the art.

[0062] The compounds of the subject invention can be formulatedaccording to known methods for preparing pharmaceutically usefulcompositions. Formulations are described in a number of sources whichare well known and readily available to those skilled in the art. Forexample, Remington's Pharmaceutical Sciencse (Martin E W [1995] EastonPa., Mack Publishing Company, 19^(th) ed.) describes formulations whichcan be used in connection with the subject invention. Formulationssuitable for administration include, for example, aqueous sterileinjection solutions, which may contain antioxidants, buffers,bacteriostats, and solutes which render the formulation isotonic withthe blood of the intended recipient; and aqueous and nonaqueous sterilesuspensions which may include suspending agents and thickening agents.The formulations may be presented in unit-dose or multi-dose containers,for example sealed ampoules and vials, and may be stored in a freezedried (lyophilized) condition requiring only the condition of thesterile liquid carrier, for example, water for injections, prior to use.Extemporaneous injection solutions and suspensions may be prepared fromsterile powder, granules, tablets, etc. It should be understood that inaddition to the ingredients particularly mentioned above, theformulations of the subject invention can include other agentsconventional in the art having regard to the type of formulation inquestion.

[0063] The cucurbitacin compound of the present invention include allhydrates and salts that can be prepared by those of skill in the art.Under conditions where the compounds of the present invention aresufficiently basic or acidic to form stable nontoxic acid or base salts,administration of the compounds as salts may be appropriate. Examples ofpharmaceutically acceptable salts are organic acid addition salts formedwith acids which form a physiological acceptable anion, for example,tosylate, methanesulfonate, acetate, citrate, malonate, tartarate,succinate, benzoate, ascorbate, alpha-ketoglutarate, andalpha-glycerophosphate. Suitable inorganic salts may also be formed,including hydrochloride, sulfate, nitrate, bicarbonate, and carbonatesalts.

[0064] Thus, the present compounds may be systemically administered,e.g., orally, in combination with a pharmaceutically acceptable vehiclesuch as an inert diluent or an assimilable edible carrier. They may beenclosed in hard or soft shell gelatin capsules, may be compressed intotablets, or may be incorporated directly with the food of the patient'sdiet. For oral therapeutic administration, the active compound may becombined with one or more excipients and used in the form of ingestibletablets, buccal tablets, troches, capsules, elixirs, suspensions,syrups, wafers, and the like.

[0065] The tablets, troches, pills, capsules, and the like may alsocontain the following: binders such as gum tragacanth, acacia, cornstarch or gelatin; excipients such as dicalcium phosphate; adisintegrating agent such as corn starch, potato starch, alginic acidand the like; a lubricant such as magnesium stearate; and a sweeteningagent such as sucrose, fructose, lactose or aspartame or aflavoringagent such as peppermint, oil of wintergreen, or cherry flavoring may beadded. When the unit dosage form is a capsule, it may contain, inaddition to materials of the above type, a liquid carrier, such asavegetable oil or a polyethylene glycol. Various other materials may bepresent as coatings or to otherwise modify the physical form of thesolid unit dosage form. For instance, tablets, pills, or capsules may becoated with gelatin, wax, shellac, or sugar and the like. A syrup orelixir may contain the active compound, sucrose or fructose as asweeting agent, methyl and propylparabens as preservatives, a dye andflavoring such as cherry or orange flavor. Of course, any material usedin preparing any unit dosage form should be pharmaceutically acceptableand substantially non-toxic in the amounts employed. In addition, theactive compound may incorporated into sustained-release preparations anddevices.

[0066] According to the method of the subject invention, cucurbitacin I,or a pharmaceutically acceptable salt or analog thereof can beadministered to a patient by itself, or co-administered with anothercompound, including cucurbitacin I, or a pharmaceutically acceptablesalt or analog thereof. Co-administration can be carried outsimultaneously (in the same or separate formulations) or consecutively.Furthermore, according to the method of the subject invention,cucurbitacin I, or a pharmaceutically acceptable salt or analog thereof,can be administered to a patient as adjunctive therapy. For example,cucurbitacin I, or a pharmaceutically acceptable salt or analog thereof,can be administered to a patient in conjunction with chemotherapy.

[0067] Thus, the cucurbitacin compounds of the subject invention(cucurbitacin I, or a pharmaceutically acceptable salt or analogthereof), whether administered separately, or as a pharmaceuticalcomposition, can include various other components as additives. Examplesof acceptable components or adjuncts which can be employed in relevantcircumstances include antioxidants, free radical scavenging agents,peptides, growth factors, antibiotics, bacteriostatic agents,immunosuppressives, anticoagulants, buffering agents, anti-inflammatoryagents, anti-pyretics, time-release binders, anesthetics, steroids, andcorticosteroids. Such components can provide additional therapeuticbenefit, act to affect the therapeutic action of the cucurbitacincompound, or act towards preventing any potential side effects which maybe posed as a result of administration of the cucurbitacin compound. Thecucurbitacin compounds of the subject invention can be conjugated to atherapeutic agent, as well.

[0068] Additional agents that can co-administered to a patient in thesame or as a separate formulation include those that modify a givenbiological response, such as immunomodulators. For example, proteinssuch as tumor necrosis factor (TNF), interferon (such asalpha-interferon and beta-interferon), nerve growth factor (NGF),platelet derived growth factor (PDGF), and tissue plasminogen activatorcan be administered. Biological response modifiers, such as lymphokines,interleukins (such as interleukin-1 (IL-1), interleukin-2 (IL-2), andinterleukin-6 (IL-6)), granulocyte macrophage colony stimulating factor(GM-CSF), granulocyte colony stimulating factor (G-CSF), or other growthfactors can be administered.

[0069] The subject invention also provides an article of manufactureuseful in treating cancer characterized by constitutive activation ofthe JAK and/or STAT signaling pathways. The article contains apharmaceutical composition containing cucurbitacin I, or apharmaceutically acceptable salt or analog thereof, and apharmaceutically acceptable carrier or diluent. The article ofmanufacture can be, for example, a vial, bottle, intravenous bag,syringe, nasal applicator, microdialysis probe, or other container forthe pharmaceutical composition. The nasal applicator containing thepharmaceutical composition of the invention can further comprise apropellent. The article of manufacture can further comprise packaging.The article of manufacture can also include printed material disclosinginstructions for concerning administration of the pharmaceuticalcomposition for the treatment of cancer. Preferably, the printedmaterial discloses instructions concerning administration of thepharmaceutical composition for the treatment of cancer characterized byconstitutive activation of the JAK/STAT signaling pathway. The printedmaterial can be embossed or imprinted on the article of manufacture andindicate the amount or concentration of the active agent (cucurbitacin Ior an analog thereof), recommended doses for treatment of the cancer, orrecommended weights of individuals to be treated.

[0070] In a further aspect, the subject invention concerns a method ofscreening substances for antitumor activity using aphosphotyrosine-STAT3-specific cytoblot. Using the method of the presentinvention, modulation of STAT3 can be utilized to evaluate the antitumorefficacy of a candidate substance on a broad spectrum of cancer cellsand cell lines.

[0071] In one embodiment, the screening method of the subject inventionis a phosphotyrosine STAT3-specific cytoblot that has allowed theidentification of the compounds described herein that exhibit JAK/STAT3signaling inhibitory activity. JSI-124 blocked activation of STAT3 inseveral human cancer cell lines that contain high levels ofconstitutively-activated tyrosine phosphorylated STAT3 and subsequentlyinhibited STAT3 DNA-binding activity and STAT3-dependent geneexpression. This JAK/STAT3 signaling disrupter is highly selective inthat other oncogenic and tumor survival pathways were not affected. Theability of JSI-124 to increase mouse survival and to inhibit growth inmice of human and murine tumors and oncogene-transformed NIH 3T3 tumorswith high levels of constitutively-activated STAT3 but not the growth ofthose with low levels of activated STAT3 further validates interferingwith STAT3 signaling as a sound approach to cancer chemotherapy. Presentstudies are geared towards evaluating the antitumor efficacy of JSI-124in a broader spectrum of human cancer cell lines and towards identifyingthe biochemical target of JSI-124.

[0072] The screening method of the subject invention involves applyingcells to a substrate, such as a tissue culture plate defining one ormore receptacles (wells) for containing the cells; contacting one ormore candidate substances with the cells; permeabilizing the cells;adding a ligand specific for STAT3 protein (preferably, forphosphotyrosine-STAT3 protein), wherein the ligand contains or isassociated with a detectable label; and detecting the label, therebydetecting phospho-STAT3 protein within the cells. Optionally, the methodcan include the step of removing or otherwise segregating any labeledligand that has not bound to, or become associated with, phospho-STAT3.The detectable label can be directly attached to the phospho-STAT3specific ligand or indirectly attached to the ligand through anintermediate entity. Optionally, the method can further include the stepof quantifying the amount of STAT3 protein in the cells. The steps ofthe method of the invention can be carried out in any appropriate orderthat permits detection of phospho-STAT3.

[0073] The substrate can be a solid support or matrix, such as amembrane. The substrate can be porous, semi-porous, or non-porous.Exemplary materials for the substrate include those utilized for thecytoblot described in Materials and Methods, as well as latex,cellulose, nitrocellulose, and nylon. Other examples of suitablesubstrate materials include polyvinylidinedifluoride (PVDF) and otherpolyvinyl materials.

[0074] Preferably, the ligand specific for phospho-STAT3 is a primaryantibody (monoclonal or polyclonal). The primary antibody can be boundor otherwise associated with the intermediate entity, such as asecondary antibody. The secondary antibody can be conjugated to adetectable label, such as horseradish peroxidase. Preferably, thedetectable label is detected by Western blot, such as a Western blotchemiluminescence assay. The amount of detected label (and hencedetected phosphotyrosine-STAT3) can be determined or quantified usingdensitometry, for example.

[0075] As used herein, the term “antibody” refers to immunoglobulinmolecules and immunologically active portions of immunoglobulinmolecules, such as molecules that contain an antigen binding site whichspecifically binds (immunoreacts with) an antigen, such asphosphorylated STAT3. Examples of immunologically active portions ofimmunoglobulin molecules include F(ab) and F(ab′)₂ fragments which canbe generated by treating the antibody with an enzyme such as pepsin.

[0076] The detectable label can operate through any of a variety ofmechanisms, permitting detection through fluorescent, luminescent,and/or enzymatic properties, for example.

[0077] Cells can be permeabilized sufficiently to allow exposure of thephospho-STAT3 ligand to phospho-STAT3 using methods and agents known tothose of ordinary skill in the art. For example, an appropriate solvent,such as methanol, can be applied to the cells.

[0078] The cells applied to the substrate can be any type of cell, suchas fibroblasts, and are preferably cancer cells of a tumor cell line.Exemplified cancer cells are disclosed herein. The cancer cells can begenetically engineered to contain an oncogene, such as src, orartificially induced to become transformed, such as by exposure to acarcinogen. Preferably, the cells applied to the substrate are cancercells, wherein the cancer is of a form characterized by constitutiveactivation of the JAK2 and/or STAT 3 signaling pathways. Preferably, thecells are whole or intact cells.

[0079] Preferred cells (transformed cells or non-transformed cells)include, for example, neurons, fibroblasts smooth muscle cells, cardiaccells, skeletal muscle cells, glial cells, embryonic stem cells, adultstem cells, mast cells, adipocytes, protozoans, bacterial cells, yeastcells, and immune cells. The cells can be vertebrate cells, includingmammalian cells, such as human or non-human mammal cells. Preferably,the cells applied to the substrate are selected from the groupconsisting of human cells, mouse cells, rat cells, and rabbit cells.

[0080] The quantity of phosphotyrosine-STAT3 detected in the cellscontacted with the candidate substance can be compared to known amountsof phosphotyrosine-STAT3 in other cells. For example, where candidatesubstances are screened by exposing cancer cells to the candidatesubstances, the quantity of phosphotyrosine-STAT3 detected in the cancercells exposed to the candidate substance can be compared to a knownquantity of phosphotyrosine-STAT3 detected in cancer cells that have notbeen exposed to the candidate substance. Further, a control step can beconducted where the quantity of phosphotyrosine-STAT3 detected in thecancer cells exposed to the candidate substance is compared to thequantity of phosphotyrosine-STAT3 detected in a corresponding normal(non-cancer) cell, where the normal cell has or has not been exposed tothe candidate agent. Comparisons of phosphotyrosine-STAT3 quantities arepreferably carried out using cells of the same type, such asfibroblasts.

[0081] Preferred substances that are tested according to the screeningmethod of the subject invention include a variety of entities, such asorganic compounds, including small molecules; lipids; carbohydrates;peptides; peptidomimetics; inorganic compounds; nucleic acids, such asDNA and RNA molecules); and ions, such as metal ions. Varioustreatments, such as radiation treatments can be applied to the testcells, as well.

[0082] Using the screening method of the subject invention, a pluralityof candidate substances, at various concentrations, can be screenedsimultaneously on a variety of different types of cells at variousconcentrations. For example, samples of various cells can be seeded intospecific wells of a substrate or solid support, such as an assay plate,and a variety of substances can be contacted to the cells, as acombination-type array. Mixtures of two or more substances can also beapplied to a single sample of cells, or multiple samples of cells.

[0083] In another aspect, the subject invention concerns substancesidentified as inhibitors of the STAT3 signaling pathway using thescreening methods disclosed herein.

[0084] In another aspect, the subject invention concerns a kit forscreening one or more substances for antitumor activity. The kit of thesubject invention can include a ligand specific forphosphotyrosine-STAT3 protein, wherein the ligand is directly orindirectly associated with a detectable label; and at least one of thefollowing: cells for screening the candidate substance(s) forphosphotyrosine-STAT3 inhibitory activity; and a substrate for applyingthe cells, as described with respect to the screening method of thesubject invention.

[0085] As used herein, the term “apoptosis”, or programmed cell death,refers to the process in which the cell undergoes a series of molecularevents leading to some or all of the following morphological changes:DNA fragmentation; chromatin condensation; nuclear envelope breakdown;and cell shrinkage.

[0086] As used herein, the term “STAT” refers to signal transducers andactivators of transcription, which represent a family of proteins that,when activated by protein tyrosine kinases in the cytoplasm of the cell,migrate to the nucleus and activate gene transcription. Examples ofmammalian STATs include STAT 1, STAT2, STAT3, STAT4, STAT5a, STAT5b, andSTAT6.

[0087] As used herein, the term “JAK” refers to a member of a family ofnon-receptor tyrosine kinases that transfers a phosphate moiety totyrosine on recipient proteins. Examples include JAK1 and JAK2.

[0088] As used herein, the term “signaling” and “signaling transduction”represents the biochemical process involving transmission ofextracellular stimuli, via cell surface receptors through a specific andsequential series of molecules, to genes in the nucleus resulting inspecific cellular responses to the stimuli.

[0089] As used herein, the term “constitutive activation,” as in theconstitutive activation of the STAT pathway, refers to a condition wherethere is an abnormally elevated level of tyrosine phosphorylated STAT3within a given cancer cell(s), as compared to a corresponding normal(non-cancer or non-transformed) cell. Constitutive activation of STAT3has been exhibited in a large variety of malignancies, including, forexample, breast carcinoma cell lines; primary breast tumor specimens;ovarian cancer cell lines and tumors; multiple myeloma tumor specimens;blood malignancies, such as acute myelogenous leukemia; and breastcarcinoma cells, as described in published PCT international applicationWO 00/44774 (Jove, R. et al.), the disclosure of which is incorporatedherein by reference in its entirety.

[0090] All patents, patent applications, provisional applications, andpublications referred to or cited herein are incorporated by referencein their entirety, including all figures and tables, to the extent theyare not inconsistent with the explicit teachings of this specification.

[0091] Materials and Methods

[0092] Cell lines. All human and murine tumor cell lines used wereobtained from American Type Culture Collection, Manassas, Va. Stablytransfected v-Src, oncogenic H-Ras, and vector NIH 3T3 cell lines havebeen described previously (Turkson, J. et al. Mol. Cell Biol., 1999,19:7519-7528; Lerner, E. C. et al. J. Biol. Chem., 1995,270:26802-26806).

[0093] Cytoblot screening for Phospho-STAT3 inhibition. NIH 3T3 cellsstably transfected with v-Src or NIH 3T3 vector control cells (Turkson,J. et al. Mol. Cell Biol., 1999, 19:7519-7528) were plated into sterile,opaque, 96-well tissue culture plates at 25,000 cells/well. Afterovernight growth at 37° C., the cells were treated for 4 hr in thepresence of either vehicle control or 10 μM of NCI Diversity Setcompounds (http://dtp.nci.nih.gov/). After treatment, cells were washedin 100 μl cold TBS (10 mM Tris, pH 7.4, 150 mM NaCl), then fixed for onehour at 4° C. with 190 μl per well of cold 3.7% formaldehyde in TBS asdescribed previously (Stockwell, B. R. et al. Chem. Biol., 1999,6:71-83). Membranes were permeabilized during a five-minute incubationin ice-cold methanol at 4° C. Cells were washed with 180 μl per well 3%milk in TBS then rocked overnight at 4° C. with 50 μl per well of 3%milk in TBS containing 1:1000 dilution of anti-phospho-STAT3 (P-Tyr 705;Cell Signaling Technology, Beverly, Mass.) and 1:2000 dilution ofHRP-conjugated goat anti-rabbit IgG (Jackson ImmunoResearch, West Grove,Pa.). Antibodies were aspirated and then plates were washed twice with180 μl per well TBS. Results were visualized by adding Western blotchemiluminescence reagent directly to the wells of the plates,incubating at room temperature for 5 min, then placing X-ray filmdirectly on top of the plate in a dark room for 1-5 min. Quantificationof results was done using GS-700 scanning densitometer (Bio-RadLaboratories, Hercules, Calif.).

[0094] Western blotting. Treated cell samples were lysed in 30 mM Hepes,pH 7.5, 10 mM NaCl, 5 mM MgCl₂, 25 mM NaF, 1 mM EGTA, 1% Triton-X-100,10% glycerol, 2 mM sodium orthovanadate, 10 μg/ml aprotinin, 10 μg/mlsoybean trypsin inhibitor, 25 μg/ml leupeptin, 2 mM PMSF, and 6.4 mg/mlp-nitrophenylphosphate. Phospho-STAT3, phospho-AKT, phospho-MEK, andphospho-p42/p44 MAPK antibodies were obtained from Cell SignalingTechnologies, Cambridge, Mass. Antibodies to STAT3, JAK2, andphospho-JNK were purchased from Santa Cruz Biotechnology, Santa Cruz,Calif. Phospho-JAK2 antibody came from Upstate Biotechnology, LakePlacid, N.Y. Membranes were blocked in either 5% milk in PBS, pH 7.4,containing 0.1% Tween-20 (PBS-T) or 1% BSA in TBS, pH 7.5, containing0.1% Tween-20 (TBS-T). Phospho-specific antibodies (excepting P-MAPK andP-JNK) were incubated in 1% BSA in TBS-T while all other antibodies werediluted in 5% milk in PBS-T for either 2 hr at room temperature orovernight at 4° C. HRP-conjugated secondary antibodies (JacksonImmunoResearch) were diluted in 5% milk in either PBS-T or TBS-T at1:1000 dilution for one hour at room temperature. Western blots werevisualized using enhanced chemiluminescence as described previously(Blaskovich, M. A. et al. Nat. Biotechnol., 2000, 18:1065-1070).

[0095] Immunoprecipitation of STAT3. A549 cells were treated for 4 hrwith vehicle or JSI-124, then lysed in 150 mM Hepes, pH 7.5, 150 mMNaCl, 1 mM EDTA, 0.5% Nonidet P-40 (NP-40), 10% glycerol, 5 mM NaF, 1 mMDTT, 1 mM PMSF, 2 mM sodium orthovanadate, and 5 μg/ml leupeptin. Samplelysates were collected and cleared, then 500 μg of lysate wasimmunoprecipitated with 50 ng STAT3 antibody overnight at 4° C. thenrocked with 25 μl Protein A/G PLUS agarose (Santa Cruz Biotechnology)for 1 hr at 4° C. Samples were washed four times with lysis buffer thenboiled in 2×SDS-PAGE sample buffer and run on 10% SDS-PAGE gel. Proteinwas transferred to nitrocellulose and then blotted as above for bothphospho-specific STAT3 and STAT3.

[0096] DNA binding and transcription. The STAT3 reporter, pLucTKS3,driving expression of firefly luciferase has been previously described(Turkson, J. et al. Mol. Cell Biol., 1999, 19:7519-7528). The pLucTKS3plasmid harbors seven copies of a sequence corresponding to theSTAT3-specific binding site in the promoter of the human C-reactiveprotein gene (Zhang, D. et al. J. Biol. Chem., 1996, 271:9503-9509). TheSTAT3-independent plasmid, pRLSRE, contains two copies of the serumresponse element (SRE) from the c-fos promoter (Yamauchi, K. et al. J.Biol. Chem., 1993, 268:14597-14600), subcloned into renilla luciferasereporter, pRL-null (Promega Corporation, Madison, Wis.).

[0097] Transfection and generation of stable clones. NIH3T3/v-Src/pLucTKS3 and NIH 3T3/v-Src/pRLSRE are stable clones that weregenerated by transfecting NIH 3T3/v-Src fibroblasts with pLucTKS3 orpRLSRE and selecting for G418-resisitant and zeocin clones, respectively(Turkson, J. et al. Mol. Cell Biol., 1999, 19:7519-7528; Turkson, J. etal. J. Biol. Chem., 2001, 28:28). In the case of NIH3T3/v-Src/pLucTKS3/pRLSRE, pRLSRE was transfected into NIH3T3/v-Src/pLucTKS3 cells and stable G418-resistant clones were selected.Transfections were carried out with LipofectAMINE Plus (InvitrogenCorporation, Carlsbad, Calif.) according to the manufacturer's protocol.Treatment of cells with inhibitors: Src-transformed NIH 3T3 cells stablyexpressing reporter constructs pLucTKS3 or pRLSRE or both were treatedwith JSI-124 (10 μM) for 24-48 hr prior to harvesting cells forcytosolic and nuclear extracts preparation and luciferase assay.

[0098] Preparation of cytosolic extracts. Cytosolic extracts wereprepared from fibroblasts as previously described (Turkson, J. et al.Mol. Cell Biol., 1999, 19:7519-7528). Briefly, after two washes with PBSand equilibration for 5 min with 0.5 ml of PBS-0.5 mM EDTA, cells werescraped off the dishes and the cell pellet was obtained bycentrifugation (4500×g, 2 min, 4° C.). Cells were resuspended in 0.4 mlof low-salt HEPES buffer (10 mM HEPES, pH 7.8, 10 mM KCl, 0.1 mM EGTA,0.1 mM EDTA, 1 mM PMSF, and 1 mM DTT) for 15 min, lysed by the additionof 20 μl of 10% NP-40, and centrifuged (10,000×g, 30 sec, 4° C.) toobtain the cytosolic supernatant, which was used for luciferase assays(Promega Corporation) measured with a luminometer.

[0099] Nuclear extract preparation and gel shift assay. Nuclear extractpreparation and electrophoretic mobility shift assay were carried out aspreviously described (Turkson, J. et al. Mol. Cell Biol., 1999,19:7519-7528). The ³²P-radiolabeled oligonucleotide probe is hSIE (highaffinity sis-inducible element, m67 variant,5′-AGCTTCATTTCCCGTAAATCCCTA-3′) (SEQ ID NO. 1) that binds STAT1 andSTAT3.

[0100] JAK Kinase Assays. A549, MDA-MB-468, and v-Src transformed NIH3T3 cells were harvested, washed three times in PBS-V (10 mM sodiumphosphate, pH 7.4, 137 mM NaCl, 1 mM sodium orthovanadate) then lysedfor 30 min on ice in JAK kinase lysis buffer (25 mM Hepes, pH 7.4, 0.1%Triton-X-100, 0.5 mM DTT, 1 mM sodium orthovanadate, 1 mM PMSF, 10 μg/mlaprotnin, 10 μg/ml leupeptin). Samples were spun at high speed to clear,and 800-1000 μg of protein was immunoprecipitated per treatmentcondition with 50 ng of either JAK1 or JAK2 antibody (Santa CruzBiotechnology) rocking overnight at 4° C. 25 μl of protein A/G PLUSagarose then was added and rocking continued for 1 hr at 4° C. Sampleswere spun to collect agarose pellet, and pellet washed twice in washbuffer (50 mM Hepes, pH 7.4, 0.1% Triton-X-100, 0.5 mM DTT, 150 mM NaCl)and once in phosphorylation buffer (50 mM Hepes, pH 7.4, 0.1%Triton-X-100, 0.5 mM DTT, 6.25 mM manganese chloride, 100 mM NaCl).Kinase reactions were performed at 30° C. for 15 min in a final volumeof 100 μl of phosphorylation buffer. Samples were pretreated with DMSOcontrol, JSI-124, and control compounds (AG490, 100 μM, and PD180790, 2μM) before addition of 20 μCi/sample γ-[³²P]ATP. Reaction was haltedusing stop buffer (wash buffer plus 10 mM EDTA), samples spun to collectpellet, then pellet washed once with stop buffer and twice with washbuffer. Samples were then placed in 2×SDS-PAGE sample buffer, boiled at100° C., and run on 8% SDS-PAGE gels to separate proteins.Autophosphorylation results were visualized by autoradiography.

[0101] Src Kinase Assay. A549, MDA-MB-468, and v-Src cells wereharvested and lysed for 30 min on ice in RIPA150 buffer (10 mM Tris, pH7.5, 150 mM NaCl, 10% glycerol, 5 mM EDTA, 1% Triton-X-100, 0.1% SDS,100 μM sodium orthovanadate, 10 μg/ml aprotinin, 10 μg/ml leupeptin, 1μg/ml antipain). Samples were spun at high speed to clear, then 1000 μgof protein were immunoprecipitated per treatment condition with 2 μgv-Src antibody (Ab-1, Oncogene Research Products, San Diego, Calif.)rocking overnight at 4° C. 25 μl of protein A/G PLUS agarose then wasadded and rocking continued for 4 hr at 4° C. Samples were spun tocollect agarose pellet, and pellet washed three times in RIPA150 buffer,twice in RIPA10 buffer (10 mM Tris, pH 7.5, 10 mM NaCl, 10% glycerol, 5mM EDTA, 1% Triton-X-100, 0.1% SDS, 100 μM sodium orthovanadate 10 μg/mlaprotinin, 10 μg/ml leupeptin, 1 μg/ml antipain), and three times in 40mM Tris, pH 7.4. Pellet was then resuspended in 30 μl kinase reactionbuffer (20 mM Tris, pH 7.4, 5 mM MgCl₂) containing 10 μCi γ-[³²P]ATP.Inhibitors were preincubated for several minutes before the addition ofthe ATP. Kinase reactions were carried out at room temperature for 15min. Reaction was stopped with the addition of 2×SDS-PAGE sample buffer.Samples were boiled and run on 10% SDS-PAGE gels. Autophosphorylationresults were visualized by autoradiography.

[0102] Antitumor activity in the nude mouse tumor xenograft model. Nudemice and C57 BL-6 black mice (National Cancer Institute, Bethesda, Md.)were maintained in accordance with the Institutional Animal Care and UseCommittee (IACUC) procedures and guidelines. v-Src- and oncogenicH-Ras-transformed NIH 3T3, A549, MDA-MB-468 and Calu-1 cells wereharvested, resuspended in PBS and injected s.c. into the right and leftflank (10×10⁶ cells per flank) of 8 week old female nude mice asreported previously (Sun, J. et al. Cancer Res., 1999, 59:4919-4926).Similarly, murine B16-F10 Melanoma cells were injected s.c. into theright and left flank (10⁶ cells per flank) of C57 black mice. Whentumors reached about 150 mm³, animals were randomized (five animals pergroup; two tumors per animal) and dosed i.p. with 0.2 ml once daily.Control animals received DMSO (20%) vehicle whereas treated animals wereinjected with JSI-124 (1 mg/kg/day) in 20% DMSO in water. The tumorvolumes were determined by measuring the length (l) and the width (w)and calculating the volume (V=lw²/2) as described previously (Sun, J. etal. Cancer Res., 1999, 59:4919-4926). Statistical significance betweencontrol and treated animals were evaluated by using Student's t-test.For the mouse survival experiments, C57 BL-6 black mice were implanteds.c. with B16-F10 cells (10⁵ to 10⁶ cells per flank). On day 5 afterimplantation the mice were randomized (6 animals per group) and treatedwith either vehicle or JSI-124 (1 mg/Kg/day) for 25 days. Percentsurviving mice was determined by monitoring the death of mice until allmice died. Two experiments of 12 animals each (6 control and 6 treatedwith JSI-124) were carried out. For statistical analysis: for each ofthe two experiments, mice receiving JSI-124 were compared to thosereceiving vehicle control with respect to survival using the permutationlog rank test as implemented in the statistical software package,ProcStatXact (p-values are two sided and exact). The results of bothexperiments were then pooled in a stratified analysis and resulted in ap-value of 0.01.

EXAMPLE 1

[0103] Development of Phosphotyrosine STAT3-Specific Cytoblot HighThroughput Assay and Identification of JSI-124

[0104] STAT3 is found tyrosine phosphorylated andconstitutively-activated in many human cancer types. Blockade of thisaberrant activation using dominant negative STAT3 was previously shownto result in inhibition of tumor growth and induction of tumor cellapoptosis, giving strong support to the validation of STAT3 as a cancerdrug discovery target (reviewed in Bowman, T. et al. Cancer Control,1999, 6:427-435; Turkson, J and R. Jove Oncogene, 2000, 19:6613-6626;and Bowman, T. et al. Oncogene, 2000, 19:2474-2488). In an attempt toidentify novel anticancer drugs based on interfering with the aberrantactivation of STAT3, a high throughput cytoblot assay was developed inwhich the levels of activated tyrosine phosphorylated STAT3 aredetermined by an antibody specific for tyrosine phosphorylated STAT3.Using this cytoblot assay, a library consisting of 1,992 compounds fromthe National Cancer Institute (referred to as the NCI Diversity Set) hasbeen screened for agents capable of blocking v-Src activation of STAT3in NIH 3T3 cells as described under Materials and Methods. Analysis ofthe cytoblot results indicated that several compounds inhibitedactivation of STAT3 to various degrees. The most potent of thesecompounds, JSI-124 (NCI identifier: NSC 521777), suppressed v-Srcactivated STAT3 at a concentration of 10 μM. FIG. 1 shows the structureof JSI-124 which is also known as cucurbitacin I (Witkowski, A. and J.Konopa Biochim. Biophys. Acta., 1981, 674:246-255; Duncan, K. L. et al.Biochem. Pharmacol, 1996, 52:1553-1560; Dinan, L. et al. Biochem. J.,1997, 327:643-650). FIG. 2 shows an example of a 96-well plate cytoblotwhere the effects of 88 compounds from the NCI Diversity Set onphosphotyrosine STAT3 levels were evaluated. JSI-124 (10 μM) at positionD6 on the plate reduced phosphotyrosine STAT3 to barely detectablelevels.

EXAMPLE 2

[0105] JSI-124 Suppresses Phosphotyrosine STAT3 Levels in Human CancerCell Lines

[0106] The results of the cytoblot shown in FIG. 2 identifies JSI-124 asan inhibitor of v-Src activation of STAT3 in NIH 3T3 cells. To determinewhether JSI-124 suppresses phosphotyrosine STAT3 levels in human cancercell lines, several human cancer cell lines were evaluated and thosewith high levels of tyrosine phosphorylated STAT3 were identified, asshown in FIG. 3A. Among the human cancer cell lines evaluated A549 (alung adenocarcinoma), MDA-MB-468 and MDA-MB-231 (two breast carcinomas),and Panc-1 (a pancreatic carcinoma) contained high levels of tyrosinephosphorylated STAT3. These human cancer cell lines along with thepositive control cell line (v-Src transformed NIH 3T3 cells) weretreated with either vehicle or JSI-124 (10 μM) for 4 hr and the celllysates processed for Western blotting with anti-phosphotyrosine STAT3antibody as described under Methods. FIG. 3B shows that JSI-124 was veryeffective at reducing the levels of tyrosine phosphorylated STAT3. Theseresults confirm those of the cytoblot and demonstrate the ability ofJSI-124 to suppress the levels of constitutively-activated, tyrosinephosphorylated STAT3 not only in v-Src transformed NIH 3T3 murinefibroblasts but also in human cancer cells of epithelial origin.

[0107] Because the phosphotyrosine STAT3 antibody could possiblycross-react with other tyrosine phosphorylated proteins, the fact thatJSI-124 suppresses phosphotyrosine STAT3 levels was confirmed by firstimmunoprecipitating STAT3 with an anti-STAT3 antibody and then Westernblotting with anti-phosphotyrosine STAT3 antibody. FIG. 3C shows thattreatment of A549 and MDA-MB-468 cells with JSI-124 (10 μM) for 4 hrreduced phosphotyrosine STAT3 to barely detectable levels. To determineif JSI-124 affected the STAT3 protein levels, reblotting with anti-STAT3antibody was carried out. FIG. 3C also shows that JSI-124 has no effecton the protein levels of STAT3. Thus, JSI-124 suppresses thephosphotyrosine levels of STAT3 without affecting its protein levels.

EXAMPLE 3

[0108] JSI-124 Inhibits STAT3 Signaling by Disrupting STAT3 DNA-BindingActivity and STAT3-Mediated Gene Expression

[0109] Tyrosine phosphorylation of STAT3 is required for its biologicalactivity (reviewed in Stark, G. R. et al. Annu. Rev. Biochem., 1998,67:227-264; Horvath, C. M. and J. E. Darnell Curr. Opin. Cell. Biol.,1997, 9:233-239, Ihle, J. N. and I. M. Kerr Trends Genet., 1995,11:69-74; Schindler, C. and J. E. Darnell Annu. Rev. Biochem., 1995,64:621-651). It was reasoned that the suppression by JSI-124 of thephosphotyrosine levels of STAT3 should lead to disruption of STAT3DNA-binding activity and STAT3-mediated gene expression. To this end,the effect of JSI-124 on STAT3 DNA-binding activity was first evaluatedby electrophoretic mobility shift assay (EMSA). v-Src transformed NIH3T3 cells and A549 cells were treated with vehicle or JSI-124 andnuclear extracts containing activated STAT3 were incubated with[³²P]-labeled hSIE oligonucleotide probe for EMSA as described underMethods. FIG. 4A shows that STAT3 DNA-binding activity was greatlyreduced in nuclear extracts from v-Src/NIH 3T3 and A549 cells treatedwith JSI-124 compared to extracts from vehicle-treated cells (lanes 4vs. 5 and 9 vs. 10). To confirm that the band seen in the gel containsSTAT3-DNA complexes, the nuclear extracts were preincubated withanti-STAT3 or anti-STAT1 antibodies. The anti-STAT3 but not anti-STAT1antibody supershifted or blocked the complex demonstrating theprotein-DNA complex contains STAT3, not STAT1 (as shown in FIG. 4A,lanes 1,2, and 3 and 6,7, and 8). These results demonstrate thatJSI-124, by reducing the levels of tyrosine phosphorylated STAT3,inhibits STAT3 signaling resulting in disruption of STAT3 DNA-binding.

[0110] It was next determined if this suppression of STAT3 activationresults in inhibition of STAT3-mediated gene expression. To this end,v-Src/NIH 3T3 fibroblasts that stably express a STAT3-dependent fireflyluciferase reporter (pLucTKS3) or that stably express a serum responseelement (SRE)-dependent renilla luciferase reporter (pRLSRE) weretreated with either vehicle or JSI-124 and cytosolic extracts preparedfor luciferase assays as described under Methods. FIG. 4B shows thatJSI-124 significantly suppresses induction of the STAT3-dependentpLucTKS3 luciferase reporter without affecting the pRLSRE reporter.Because in v-Src transformed NIH 3T3 cells, v-Src activates pLucTKS3 ina STAT3-dependent manner and pRLSRE in a STAT3-independent manner, theresults shown in FIG. 4B demonstrate that JSI-124 is specific toSTAT3-mediated transcription. Thus, JSI-124 inhibits STAT3 signaling bysuppressing phosphotyrosine levels of STAT3, inhibiting STAT3-DNAbinding and STAT3-mediated gene expression.

EXAMPLE 4

[0111] JSI-124 Suppresses Phosphotyrosine Levels of STAT3 and JAK2 ButNot Src in A549 and MDA-MB-468 Cells

[0112] The ability of JSI-124 to suppress phosphotyrosine levels ofSTAT3 suggests that this agent may interfere with the function of theupstream tyrosine kinases JAK and Src that are known to phosphorylateSTAT3. The effects of JSI-124 on the phosphotyrosine levels of JAK2 andSrc in whole cells as well as the ability of JSI-124 to inhibit Src,JAK1, and JAK2 kinase activities in vitro were evaluated. FIG. 5A showsthat treatment of A549 and MDA-MB-468 with JSI-124 results in reductionof the levels of tyrosine phosphorylated STAT3, with A549 cells beingmore sensitive than MDA-MB-468. Furthermore, JSI-124 was also effectiveat suppressing the levels of tyrosine phosphorylated JAK2 but not thoseof tyrosine phosphorylated Src. JSI-124 had no effect on the proteinlevels of STAT3 and JAK2 in both cell lines, as shown in FIG. 5A.

[0113] The effects of JSI-124 on JAK/STAT3 signaling described abovewere determined after 4 hr of JSI-124 treatment. To ascertain the lengthof treatment time required for JSI-124 to suppress phosphotyrosine STAT3and JAK levels, a time course experiment was carried out. FIG. 5B showsthat treatment of A549 and MDA-MB-468 cells with JSI-124 for as littleas 60 min was effective, and in both cell lines the suppression wascomplete by 2 hr. Thus, the suppression by JSI-124 of the levels oftyrosine phosphorylated STAT3 and JAK2 is rapid.

[0114] The ability of JSI-124 to inhibit the kinase activities of Src,JAK1, and JAK2 in vitro was next evaluated. To this end, Src, JAK1, andJAK2 were immunoprecipitated from either A549, MDA-MB-468, or v-Src/NIH3T3 cells and incubated the immunoprecipitates with either vehiclecontrol, JSI-124, the JAK tyrosine kinase inhibitor AG490, or the Srckinase inhibitor PD180970 and followed autophosphorylation of Src, JAK1,and JAK2 as described under Methods. FIG. 5C shows that in all threecell lines as expected PD180970 inhibits Src but not JAK1 or JAK2activities (A549 did not have JAK1 kinase activity). Similarly, AG490inhibited JAK1 and JAK2 but not Src kinase activities. In contrast, allthree kinase activities were not affected by JSI-124, as shown in FIG.5C. Therefore, although in whole cells JSI-124 is very effective atsuppressing the levels of tyrosine phosphorylated STAT3 and JAK2, it isunable to inhibit directly Src, JAK1, or JAK2 kinase activities invitro.

EXAMPLE 5

[0115] JSI-124 is Highly Selective for the JAK/STAT3 Over Akt, Erk, andJNK Signaling Pathways

[0116] To determine whether the effects of JSI-124 were selective to theJAK/STAT3 pathway over other oncogenic and survival pathways, A549 andMDA-MB-468 cells were treated with various concentrations of JSI-124 andprocessed the lysates for Western blotting with antibodies specific forphospho-STAT3, phospho-Erk1/2, phospho-JNK, and phospho-Akt as describedunder Methods. FIG. 6 shows that A549 and MDA-MB-468 have constitutivelyphosphorylated Erk1/Erk2, JNK1, and Akt in addition to phospho-STAT3.Treatment with JSI-124 resulted in suppression of phospho-STAT3 levelsin both cell lines. In contrast, treatment with JSI-124 had noinhibitory effect on phospho-Akt, phospho-Erk1/2, or phospho-JNK with aconcentration as high as 10 μM. With Erk1/2, not only did JSI-124 notinhibit but actually it increased the levels of phosphorylation. Thus,these results demonstrate that JSI-124 suppressive effects are highlyselective for the JAK/STAT3 over Erk, JNK, and Akt tumor survival andoncogenic signaling pathways.

EXAMPLE 6

[0117] JSI-124 Inhibits Growth in Mice of Tumors with High Levels ofConstitutively-Activated STAT3

[0118] Previous studies have shown that interfering with STAT3 signalingusing a gene therapy approach with a dominant negative variant of STAT3(STAT3-β) resulted in inhibition of the growth of melanoma cells in nudemice (Niu, G. et al. Cancer Res., 1999, 59:5059-5063; Catlett-Falcone,R. et al. Immunity, 1999, 10:105-115). Because JSI-124 inhibitsaberrantly activated STAT3 signaling, DNA binding, and STAT3-mediatedgene expression, it was reasoned that the growth in nude mice of tumorswith constitutively-activated STAT3 should be more sensitive to JSI-124than that of tumors with low or without constitutively-activated STAT3.To this end, A549 and MDA-MB-468 as well as Calu-1 cells, a lungadenocarcinoma which has barely detectable levels of tyrosinephosphorylated STAT3 (FIG. 3A), were implanted subcutaneously in nudemice. When the tumors reached an average size of about 150 mm³, theanimals were randomized and treated intraperitoneally with eithervehicle or JSI-124 (1 mg/kg/day) as described under Methods. FIGS. 7Band 7C, respectively, show that A549 and Calu-1 tumors from animalstreated with vehicle grew to about 500 mm³ twenty-six days after tumorimplantation. MDA-MB-468 treated with vehicle control grew to about 300mm³ sixty days after tumor implantation, as shown in FIG. 7A. FIGS. 7Band 7A show that JSI-124 inhibited A549 and MDA-MB-468 tumor growth by76% and 86%, respectively. In contrast, JSI-124 had little effect on thegrowth in nude mice of Calu-1 cells, as shown in FIG. 7C. Treatment ofmice bearing A549 cells with a reduced dose of 0.5 mg/kg/day for 23 daysalso inhibited tumor growth by 52% (data not shown). At both doses, 1mg/kg/day and 0.5 mg/kg/day, JSI-124 had no effects on body weight,activity or food intake of mice. However, at the local site of druginjection, the peritoneal cavity, JSI-124 at the 1 mg/kg/day dose,caused edema. A similar observation was made by the National CancerInstitute Developmental Therapeutics Program where edema was observed atthe subcutaneous site of injection (Jill Johnson, NCI, personalcommunication).

[0119] The results from A549, Calu-1, and MDA-MB-468 xenograft studiessuggest that human cancer cells which express constitutively-activatedSTAT3 should be sensitive to JSI-124. It was further reasoned that ifthe ability of JSI-124 to inhibit tumor growth in nude mice depends onconstitutively-activated STAT3, v-Src-transformed NIH 3T3 cells whichrequire constitutively-activated STAT3 for malignant transformation (Yu,C. L. et al. Science, 1995, 269:81-83) should be sensitive to JSI-124whereas oncogenic Ras-transformed NIH 3T3 cells, where STAT3 is notconstitutively-activated (see FIG. 3A), should be resistant. FIGS. 7Dand 7F show that, in the absence of JSI-124, the growth of both v-Src-and Ras-transformed NIH 3T3 tumors was highly aggressive and reachedaverage sizes of about 2500 mm³ and 1000 mm³, respectively, within 9days of tumor cell implantation. FIG. 7D also shows that JSI-124 (1mg/kg/day) inhibited the growth of v-Src/NIH 3T3 tumors by 64%. Incontrast, the growth of Ras/NIH 3T3 tumors was resistant to JSI-124, asshown in FIG. 7F. These results coupled with those from the human tumorxenografts show that JSI-124 selectively targets tumors withconstitutively-activated STAT3 signaling.

[0120] The above results were obtained from experiments withimmune-deficient nude mice. Furthermore, the studies did not investigatethe effects of JSI-124 on the survival of mice bearing death-inducingtumors. Therefore, the ability of JSI-124 to inhibit tumor growth andincrease survival of immunologically-competent C57 black mice s.c.implanted with the murine B16-F10 melanoma that expresses constitutivelyactivated STAT3 (Niu, G. et al. Cancer Res., 1999, 59:5059-5063) wasevaluated. FIG. 6 shows that B16-F10 tumors from control mice injectedwith vehicle grew to an average size of 1194±141 whereas those treatedwith JSI-124 (1 mg/Kg/day) grew only to an average size of 588±94. Thus,JSI-124 treatment inhibited tumor growth by 56%. To determine the effectof JSI-124 on mouse survival, s.c. B16-F10 melanoma was implanted andmice survival was followed over time. FIG. 6 shows that mice treatedwith vehicle begin to die on day 19 after B16-F10 implantation. By day21, half of the mice were dead, and by day 35 all 6 mice were dead. Incontrast, none of the JSI-124-treated mice were dead by day 23, half ofthe mice died on day 34 and all the mice died by day 42. FIG. 6 alsoshows that 50% of vehicle-treated mice survived up until day 21 (T₅₀=21)whereas the JSI-124-treated group of mice had a longer T₅₀ of 34 days.Thus, treatment with JSI-124 significantly increased the life span (T₅₀increase of 13 days) of immunologically-competent mice implanted withB16-F10 melanoma.

EXAMPLE 7

[0121] Cucurbitacin Analogs

[0122] The identification from the NCI diversity set of cucurbitacin I(JSI-124) as a potent (IC₅₀=200 nM) suppressor of the JAK/STAT3 tumorsurvival pathway with potent antitumor activity. Structure-activityrelationship studies were then carried out in order to identify furtherantitumor agents representing analogs of cucurbitacin I. To this end, 6analogs of cucurbitacin I were received from NCI and their ability toinhibit the levels of phosphotyrosine JAK2 and phosphotyrosine STAT3 inthe human lung carcinoma cell line A-549 were evaluated by Westernblotting as described in Materials and Methods with respect tocucurbitacin I. FIG. 15 shows representative blots of the effects ofcucurbitacins A, B, D, E, I, Tet I and Q (0-10 μM) on the levels ofphospho STAT3 and phospho JAK2 in A-549 cells. FIG. 17 shows the averageand standard deviation of at least three independent experiments foreach cucurbitacin molecule tested. From the SAR studies, the followingconclusions were made.

[0123] Comparing cucurbitacin I to cucurbitacin E (the structures ofwhich are shown in FIG. 1 and FIG. 12, respectively) reveals that ahydroxyl group of cucurbitacin I is important for phosphotyrosine-STAT3but not phosphotyrosine-JAK2 activity, as its replacement with an acetylgroup resulted in a four-fold loss of activity against phospho-STAT3without affecting activity against JAK2 (IC₅₀ for phospho-STAT3, 200 nMfor I; 800 nM for E; IC₅₀ for phospho-JAK2, 200 nM for I; 200 nM for E).

[0124] Comparing cucurbitacin B and cucurbitacin E (shown in FIG. 10 andFIG. 12, respectively) shows that reducing a carbon-carbon double bondresults in an additional five-fold loss of inhibitory activity againstphosphotyrosine-STAT3, and no reduction in activity againstphosphotyrosine-JAK2 (IC₅₀ for phospho-STAT3, 4 μM for B; IC₅₀ for JAK2,200 nM for B).

[0125] Conversion of a carbonyl group in cucurbitacin B (shown in FIG.10) to a hydroxyl, as in cucurbitacin Q (shown in FIG. 13), results inadditional loss of activity against JAK2 activation, with 2-fold gain inactivity against STAT3 activation (IC₅₀ for phospho-STAT3, 2 μM for Q;IC₅₀ for phospho-JAK2, >10 μM for Q).

[0126] The addition of a hydroxyl group in cucurbitacin B (shown in FIG.10) to get cucurbitacin A (shown in FIG. 9) results in a loss ofactivity against phosphotyrosine-STAT3 activity, as well as a decreaseof the ability of the compound to inhibit phosphotyrosine JAK2 (IC50 forphospho-STAT3, >10 μM for A; IC50 for phospho-JAK2, 1 μM).

[0127] Replacement of an acetate in cucurbitacin B (shown in FIG. 10) bya hydroxyl yields cucurbitacin D (shown in FIG. 11) and reduces activityagainst phospho STAT3 by 2-fold with no effects on phospho JAK2.

[0128] Reduction of two double bonds in cucurbitacin I (shown in FIG. 1)yields tetrahydro cucurbitacin (shown in FIG. 14) and results inreduction of activity against phospho-STAT3 and phospho JAK2.

[0129] The ability of the cucurbitacin analogs (at 1 mg/Kg/dayintraperitonealy) to inhibit the growth of v-src transformed NIH 3T3cells implanted subcutaneously in nude mice was evaluated, as describedwith respect to cucurbitacin I. FIG. 17 shows that cucurbitacin Q,tetrahydro-cucurbitacin I, cucurbitacin I, and cucurbitacin E, eachinhibit tumor growth. Cucurbitacin A inhibited tumor growth to a lesserextent (160%). Cucurbitacin B was toxic at 1 mpk. However, at 0.2mpk/day, cucurbitacin B was not toxic and inhibited tumor growth by 40%.Next, the ability of the analogs to inhibit phospho STAT3 or phosphoJAK2 levels was correlated to their ability to inhibit tumor growth innude mice. FIG. 16 shows that cucurbitacin I, which inhibits bothphospho STAT3 and phospho JAK2 levels (see inset), inhibit tumor growth.In contrast, cucurbitacin A, which inhibit only phospho JAK2 but notphospho STAT3 levels (see inset), did not significantly inhibit tumorgrowth. Furthermore, cucurbitacin Q, which inhibits phospho STAT3 butnot phospho JAK2 levels (see inset), was as potent as curcubatacin I atinhibiting tumor growth. These results indicate that a cucurbitacin withthe ability to inhibit STAT3 activation alone (cucurbitacin Q) issufficient to inhibit tumor growth. However, cucurbitacin A, whichinhibits only JAK2 activation, inhibits tumor growth to a lesser extent.Also, the ability of cucurbitacin I to inhibit both JAK2 and STAT3activation does not make it a better inhibitor of tumor growth than thecucurbitacin that inhibits STAT3 alone. This indicates that, in a modelwhere both JAK2 and STAT3 are actively signaling cells to grow andsurvive, it may be more important to shut down the constitutivesignaling of STAT3.

[0130] It should be understood that the examples and embodimentsdescribed herein are for illustrative purposes only and that variousmodifications or changes in light thereof will be suggested to personsskilled in the art and are to be included within the spirit and purviewof this application.

1 1 1 24 DNA Artificial Sequence Oligonucleotide probe 1 agcttcatttcccgtaaatc ccta 24

We claim:
 1. A method for treating cancer comprising administeringcucurbitacin I, or a pharmaceutically acceptable salt or analog thereof,to a patient in need of such treatment.
 2. The method according to claim1, wherein the analog is a compound of structure I, or apharmaceutically acceptable salt thereof:

wherein R₁, R₂, R₃, R₄, R₅, and R₆ can each be the same or different,and are each selected from the group consisting of H, O, hydroxyl,alkyl, alkenyl, alkynyl, halogen, alkoxy, aryl and heteroaryl.
 3. Themethod according to claim 1, wherein the analog is selected from thegroup consisting of cucurbitacin A, cucurbitacin B, cucurbitacin D,cucurbitacin E, cucurbitacin Q, and tetrahydro-cucurbitacin I.
 4. Themethod according to claim 1, wherein the cucurbitacin I, or apharmaceutically acceptable salt or analog thereof, inhibits the JAK2signaling pathway, the STAT3 signaling pathway, or both the JAK2 andSTAT3 signaling pathways.
 5. The method according to claim 1, whereinthe analog inhibits the STAT3 signaling pathway, but does not inhibitthe JAK2 signaling pathway.
 6. The method according to claim 1, whereinthe cucurbitacin I, or a pharmaceutically acceptable salt or analogthereof, inhibits both the JAK2 and STAT3 signaling pathways.
 7. Themethod according to claim 1, wherein the cancer is characterized byabnormal STAT3 pathway activity.
 8. The method according to claim 1,wherein the cancer is characterized by abnormal JAK2 pathway activityand abnormal STAT3 pathway activity.
 9. The method according to claim 1,wherein the patient is suffering from a tumor and the compound inhibitsgrowth of the tumor.
 10. The method according to claim 9, wherein thecompound inhibits growth of the tumor.
 11. The method according to claim1, wherein the cancer is selected from the group consisting of lungcancer, pancreatic cancer, colon cancer, ovarian cancer, and breastcancer.
 12. The method according to claim 1, wherein cucurbitacin I, ora pharmaceutically acceptable salt thereof, is administered to thepatient.
 13. The method according to claim 1, wherein the route of saidadministration is selected from the group consisting of intravenous,intramuscular, oral, and intra-nasal.
 14. A pharmaceutical compositioncomprising cucurbitacin I, or a pharmaceutically acceptable salt oranalog thereof, and a pharmaceutically acceptable carrier or diluent.15. The pharmaceutical composition of claim 14, wherein said analog is acompound of structure I, or a pharmaceutically acceptable salt thereof:

wherein R₁, R₂, R₃, R₄, R₅, and R₆ can each be the same or different,and are each selected from the group consisting of H, O, hydroxyl,alkyl, alkenyl, alkynyl, halogen, alkoxy, aryl and heteroaryl.
 16. Thepharmaceutical composition of claim 14, wherein said analog is selectedfrom the group consisting of cucurbitacin A, cucurbitacin B,cucurbitacin, D, cucurbitacin E, cucurbitacin Q, andtetrahydro-cucurbitacin I.
 17. The pharmaceutical composition of claim14, wherein the composition further comprises an immunomodulating agent.18. The pharmaceutical composition of claim 14, wherein the compositionfurther comprises an agent selected from the group consisting of anantioxidant, free radical scavenging agent, peptide, growth factor,antibiotic, bacteriostatic agent, immunosuppressive, anticoagulant,buffering agent, anti-inflammatory agent, anti-pyretic, time-releasebinder, anesthetic, steroid, and corticosteroid.
 19. An article ofmanufacture useful in treating cancer, said article containing apharmaceutical composition comprising cucurbitacin I, or apharmaceutically acceptable salt or analog thereof, and apharmaceutically acceptable carrier or diluent.
 20. The article ofmanufacture of claim 19, wherein said analog is a compound of structureI, or a pharmaceutically acceptable salt thereof:

wherein R₁, R₂, R₃, R₄, R₅, and R₆ can each be the same or different,and are each selected from the group consisting of H, O, hydroxyl,alkyl, alkenyl, alkynyl, halogen, alkoxy, aryl and heteroaryl.
 21. Thearticle of manufacture of claim 19, wherein said analog is selected fromthe group consisting of cucurbitacin A, cucurbitacin B, cucurbitacin, D,cucurbitacin E, cucurbitacin Q, and tetrahydro-cucurbitacin I.
 22. Thearticle of manufacture of claim 19, wherein the article furthercomprises an immunomodulating agent.
 23. The article of manufacture ofclaim 19, wherein the article further comprises an agent selected fromthe group consisting of an antioxidant, free radical scavenging agent,peptide, growth factor, antibiotic, bacteriostatic agent,immunosuppressive, anticoagulant, buffering agent, anti-inflammatoryagent, anti-pyretic, time-release binder, anesthetic, steroid, andcorticosteroid.
 24. The article of manufacture of claim 19, wherein thearticle is selected from the group consisting of an intravenous bag, avial, a syringe, a nasal applicator, and a microdialysis probe.
 25. Thearticle of manufacture of claim 19, wherein the article furthercomprises printed matter disclosing instructions for administering saidpharmaceutical composition for the treatment of cancer.
 26. The articleof manufacture of claim 19, wherein the article further comprisespackaging.
 27. A method of screening a substance for anti-tumoractivity, said method comprising: a. applying cells to a substrate; b.contacting the cells with one or more candidate substances; c.permeabilizing the cells; d. contacting a ligand specific forphosphotyrosine-STAT3 protein with the cells, wherein the ligand isdirectly or indirectly associated with a detectable label; and e.detecting the label, thereby detecting the presence ofphosphotyrosine-STAT3 protein within the cells.
 28. The method accordingto claim 27, wherein said method further comprises quantifying theamount of phosphotyrosine-STAT3 protein in the cells.
 29. The methodaccording to claim 27, wherein the ligand specific forphosphotyrosine-STAT3 protein is a first antibody, or an immunoreactivefragment thereof, and wherein the method further comprises contacting asecond antibody, or an immunoreactive fragment thereof, with the firstantibody, wherein the second antibody is specific for the firstantibody, and wherein the detectable label is associated with the secondantibody.
 30. The method according to claim 27, said method furthercomprising comparing the amount of phosphotyrosine-STAT3 proteindetected within the cancer cells to a known amount ofphosphotyrosine-STAT3 protein contained within cancer cells that havenot been exposed to the substance.
 31. The method according to claim 27,said method further comprising comparing the amount ofphosphotyrosine-STAT3 protein detected within the cancer cells to aknown amount of phosphotyrosine-STAT3 protein within a non-cancer cell.32. The method according to claim 27, wherein the candidate substance isa compound selected from the group consisting of an organic moiety,inorganic moiety, peptide, peptidomimetic, nucleic acid, carbohydrate,and lipid, or a combination thereof.
 33. The method according to claim27, wherein the cells applied to the substrate are cancer cells.
 34. Themethod according to claim 27, wherein the cells applied to the substrateare cancer cells from a tumor cell line.
 35. The method according toclaim 27, wherein the cells applied to the substrate are mammaliancells.
 36. The method according to claim 27, wherein the cells appliedto the substrate are selected from the group consisting of human cells,mouse cells, rat cells, and rabbit cells.
 37. A kit for screening one ormore substances for antitumor activity comprising a ligand specific forphosphotyrosine-STAT3 protein, wherein the ligand is directly orindirectly associated with a detectable label; and at least one of thefollowing: a. cells for screening the candidate substances forphosphotyrosine-STAT3 inhibitory activity; and b. a substrate forapplying the cells.
 38. The kit of claim 37, wherein the kit comprisingboth cells for screening the candidate substances forphosphotyrosine-STAT3 inhibitory activity and a substrate for applyingthe cells.
 39. The kit of claim 37, wherein said ligand is an antibody,or an immunoreactive fragment thereof.